A nanocompartment system contributes to defense against oxidative stress in Mycobacterium tuberculosis

Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, United States
Department of Molecular and Cell Biology, Division of Biochemistry, Biophysics and Structural Biology, University of California, Berkeley, United States
Department of Plant and Microbial Biology, University of California, Berkeley, United States
Department of Microbiology and Immunology, The University of British Columbia, Canada
School of Public Health, Division of Infectious Diseases and Vaccinology, University of California, Berkeley, United States

Nov 9, 2021

https://doi.org/10.7554/eLife.74358

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Accepted for publication after peer review and revision.

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Version of Record published: December 1, 2021 (This version)
Accepted Manuscript published: November 9, 2021 (Go to version)
Accepted: November 6, 2021
Received: September 30, 2021
Preprint posted: August 31, 2020 (Go to version)

Figures
Tables
Additional files

4 figures, 1 table and 20 additional files

Figures

Figure 1 with 1 supplement

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Mtb produces endogenous nanocompartments that package a peroxidase.

(A) Schematic of the nanocompartment operon in Mtb that encodes the encapsulin shell protein (Cfp29) and the dye-decoloring peroxidase cargo protein (DyP). (B) Transmission electron microscopy (TEM) of Cfp29 encapsulin proteins purified following heterologous expression of the Mtb nanocompartment operon in *E. coli*. (C) Size distribution of Cfp29 protomers purified from *E. coli*. (D) Peptide counts from mass spectrometry analysis of endogenous nanocompartments purified from Mtb. (E) TEM of endogenous nanocompartments purified from Mtb. Peroxidase activity of (F) unencapsulated and (G) encapsulated DyP (5 nM) using ABTS (480 nM) as a substrate in the presence of H₂O₂ (480 nM) at varying pH levels (4.0–6.0) as reported by a change in the absorbance at 420 nm. neg, no added enzyme.

Figure 1—figure supplement 1

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Nanocompartment purification and complementation strategies.

(A) Coomassie-stained SDS-PAGE of fractions collected during purification of nanocompartments heterologously expressed in *E. coli*: (1) ultracentrifugation pellet post-CellLytic B solubilization, (2) size-exclusion chromatography input, (3) lane 1 diluted, (4) lane 2 diluted, and (5) encapsulin fraction from size exclusion. (B) Coomassie-stained SDS-PAGE of sucrose fractions collected during purification of nanocompartments from wild-type Mtb lysates: (1) fraction containing assembled encapsulin nanocompartment complexes, (5) ladder (6), lane 1 boiled in SDS for 30 min to dissociate encapsulin nanocompartment into monomers. (C) DyP samples with or without addition of hemin were analyzed by SDS-PAGE. Samples were loaded either in their unboiled native state (left half) or heat-denatured by boiling at 95°C for 15 min. Addition of hemin yields a tetrameric DyP at 144 kDa. (D) Western blot for Cfp29 (arrow) and Ag85A control (star) from wild-type Mtb (lane 1) and Δoperon mutant (lane 2) lysates. (E) Complementation strategy schematic for *DyP::Tn* mutants (top). *DyP::Tn* mutants were transformed with ATc-inducible complementation constructs encoding the unencapsulated cargo protein (DyP), the encapsulin shell protein (Cfp29), or the nanocompartment operon (Operon). Lysates from each strain were used for nanocompartment purification. Sucrose fractions containing high molecular weight Cfp29 protomers were identified in complemented strains expressing the encapsulin shell and the operon (middle) and were analyzed using TEM (bottom).

Figure 2

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Nanocompartments protect Mtb from oxidative stress in acidic environments.

(A) OD₆₀₀ measurements of wild-type and *DyP::Tn* Mtb grown in 7H9 medium following exposure to H₂O₂ for 96 hr. Values reported are normalized to the untreated controls. CFU enumeration of wild-type and Mtb nanocompartment mutants grown in (B) standard 7H9 medium (pH 6.5) and (**C–F**) acidified 7H9 medium (pH 4.5) following exposure to oxidative stress (2.5 mM H₂O₂) for 72 hr. (G) Fluorescence emissions of wild-type and Δoperon Mtb expressing mrx1-roGFP exposed to 5 mM H₂O₂ at pH 4.5 in 7H9 medium for 60 min. Data are reported as a ratio of fluorescence emissions following excitation at 490 nm and 390 nm. Figures are representative of at least two (**E, F**) or three (**A–D, G**) independent experiments. p-Values were determined using unpaired t-test. *p<0.05, **p<0.01.

Figure 3

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Susceptibility of Mtb nanocompartment mutants to oxidative and acid stress is mediated by free fatty acids.

(A) Transposon sequencing (Tn-seq) data showing normalized sequence reads per gene for all putative Mtb peroxidases, catalases, and superoxide dismutases and (B) lipid and cholesterol metabolism Mtb mutants that were significantly attenuated following 72 hr exposure to 2.5 mM H₂O₂ at pH 4.5. (C) CFU enumeration of wild-type Mtb and Δoperon mutants following 24 hr exposure to 2.5 mM H₂O₂ at pH 4.5 in Sauton’s minimal medium and (D) 72 hr exposure to 2.5 mM H₂O₂ at pH 4.5 in 7H9 medium prepared using fatty acid (FA)-free bovine serum albumin (BSA) ± oleic acid (150 µM). (E) Intrabacterial pH measurements of wild-type and Δoperon Mtb expressing pUV15-pHGFP following 20 min exposure to 5 mM H₂O₂ at pH 6.5 or pH 4.5. 7H9 medium was prepared with standard BSA or FA-free BSA. Figures are representative of at least two (D) or three (**A–C, E**) independent experiments. p-Values were determined using an unpaired t-test. *p<0.05, **p<0.01.

Figure 4

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Nanocompartment mutants are attenuated for survival in macrophages and are more susceptible to pyrazinamide (PZA) treatment.

CFU enumeration of wild-type Mtb and (A) Δoperon or (B) DyP::Tn mutants during infection of murine bone marrow-derived macrophages. Macrophages were infected with a bacterial MOI of 1, and CFUs were enumerated immediately following phagocytosis and at days 2 and 4. Error bars are SD from four replicate wells. (C) CFU enumeration of wild-type and Mtb Δoperon mutants following 72 hr exposure to PZA (24 μg/mL) and H₂O₂ (2.5 mM) in acidified 7H9 medium (pH 5.5). Comp = *Δoperon + pOperon*. (D) Infection of BALB/C mice with WT and Δoperon mutant with and without treatment with 150 mg/kg PZA. CFU at 35 days post infection in the lung is shown. (A) and (B) are representative of at least five independent experiments; (C) is representative of three experiments and (D) is representative of two experiments. p-Values were determined using an unpaired t-test. **p<0.01; ***p<0.001.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (Mycobacterium tuberculosis)	Dyp	GenBank	Gene ID: 885388, Rv0799c
Gene (My. tuberculosis)	Cfp29	GenBank	Gene ID: 885460, Rv0798c
Strain, strain background (Mus musculus)	BALB/C	The Jackson Laboratory	Stock no: 000651
Strain, strain background (M. tuberculosis)	H37Rv	Eric Rubin Lab, Harvard School of Public Health
Strain, strain background (Escherichia coli)	BL21 (DE3) LOBSTR	kerafast	Cat# EC1002
Genetic reagent (M. tuberculosis)	DyP::Tn	Broad Institute, Hung Lab	Rv0799c::Tn
Genetic reagent (M. tuberculosis)	Δoperon	This study	ΔRv0799c-Rv0798c	See Materials and methods
Genetic reagent (M. tuberculosis)	ΔCfp29	This study	ΔRv0798c	See Materials and methods
Genetic reagent (M. tuberculosis)	ΔDyP	This study	ΔRv0799c	See Materials and methods
Recombinant DNA reagent	Pet14b	Novagen	Cat# 69660-3
Recombinant DNA reagent	pUV15tetORm	Addgene	Cat# 17975	AHT-inducible construct for all complementation
Recombinant DNA reagent	pKL4	This study		Rv0798c cloned into pUV15tetORm (with KanR)
Recombinant DNA reagent	pKL5	This study		Rv0799c cloned into pUV15tetORm (with KanR)
Recombinant DNA reagent	pKL6	This study		Operon cloned into pUV15tetORm (with KanR)
Recombinant DNA reagent	pKL14	This study		Operon cloned into pUV15tetORm (with HygR)
Recombinant DNA reagent	pKL15	This study		Rv0798c cloned into pUV15tetORm (with HygR)
Recombinant DNA reagent	pKL16	This study		Rv0799c cloned into pUV15tetORm (with HygR)
Recombinant DNA reagent	pUV15 pHGFP HygR:	Addgene	Cat# 70045	Rv0799c cloned into pUV15tetORm (with HygR)
Recombinant DNA reagent	pMV762-mrx1-roGFP2	Amit Singh, ICGEB, India		PMC3907381
Antibody	Anti-Mtb Cfp29	Rabbit polyclonal	Produced by GenScript USA, see Materials and methods	1:10,000
Antibody	HRP	Goat anti-rabbit polyclonal	Santa Cruz Biotechnology sc-2030	1:5000
Chemical compound, drug	3-Ethylbenzothia zoline-6-sulfonic acid	Millipore Sigma	Cat# 10102946001